|Year : 2016 | Volume
| Issue : 2 | Page : 52-56
Vascular endothelial growth factor expression in oral cancer and its role as a predictive marker: A prospective study
Ashish Singhal1, Rahat Hadi2, Arun Chaturvedi1, ID Sharma3, Sanjeev Misra4, Nuzhat Husain5
1 Department of Surgical Oncology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Radiation Oncology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
3 Department of Surgical Oncology, All India Institute of Medical Sciences, Jodhpur, Rajasthan, India
4 Department of Surgical Oncology, King George's Medical University, Lucknow, Uttar Pradesh, India
5 Department of Pathology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
|Date of Web Publication||8-Jun-2016|
Department of Surgical Oncology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Vibhuti Khand, Gomti Nagar, Lucknow, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Oral cancer is one of the common cancers in India with dismal survival in advanced stages. Most of the patients present in advanced stages with borderline operability and such patients may be helped by chemotherapy to render them operable. It is well known that tumor growth is angiogenesis-dependent and thus vascular endothelial growth factor (VEGF) may be a surrogate marker of growth and angiogenesis. At present, there is a scarcity of predictive markers for oral cancer. In this prospective study, we studied VEGF expression and its role as a predictive marker in oral cancer. Materials and Methods: Patients with locally advanced oral cancer having borderline operability or unfit to undergo surgery in the primary setting were included in the study. VEGF expression of the cancerous tissue was studied in all patients. Three cycles of neoadjuvant chemotherapy (NACT) was administered before definitive treatment in locally advanced cases and response is assessed. VEGF levels were analyzed in cancer tissue and compared with normal surrounding mucosa. The response to chemotherapy was then correlated with VEGF score in patients receiving NACT to evaluate it as a predictive marker. Results: All forty patients were VEGF-positive and had a mean score of 1023 with 63% patients having Grade 3 expressions, whereas the normal surrounding mucosa had a VEGF score of 30. It was seen that in patients showing no response to treatment, the mean total VEGF score was significantly higher as compared to those showing complete or partial response. VEGF score correlated inversely with chemotherapy response, but no significant association was seen between VEGF grade and chemotherapy response. Conclusion: We can conclude from this study that VEGF is significantly overexpressed in cancer mucosa as compared to normal mucosa and overexpression of VEGF was found to be associated with chemoresistance and thus may serve as a negative predictive marker.
Keywords: Chemotherapy, oral cancer, radiotherapy, surgery, vascular endothelial growth factor
|How to cite this article:|
Singhal A, Hadi R, Chaturvedi A, Sharma I D, Misra S, Husain N. Vascular endothelial growth factor expression in oral cancer and its role as a predictive marker: A prospective study. Saudi Surg J 2016;4:52-6
|How to cite this URL:|
Singhal A, Hadi R, Chaturvedi A, Sharma I D, Misra S, Husain N. Vascular endothelial growth factor expression in oral cancer and its role as a predictive marker: A prospective study. Saudi Surg J [serial online] 2016 [cited 2020 May 31];4:52-6. Available from: http://www.saudisurgj.org/text.asp?2016/4/2/52/183673
| Introduction|| |
Oral cancer is considered as a relatively chemoresistant tumor with an objective response rate of 20-30% to single agent, 40-60% to combination chemotherapy with a complete response in 30-40%.  At present, no definitive predictive marker is studied in large prospective trials for estimating the response to chemotherapy in oral cancer. Some proposed includes epidermal growth factor receptor, p53, cyclin D1, Bcl-2, proliferating cell nuclear antigen expression, and microvessel density.  Overexpression of p53 and cyclin D1 correlates with good response to neoadjuvant treatment with cisplatin and 5-fluorouracil. , Overexpression of Bcl-1 confers cytotoxic drug resistance by its ability to inhibit chemo-induced apoptosis.  Reduced Bcl-xL expression was associated with complete response. 
Others include histological aggressive pattern of invasion which is associated with improved organ preservation with chemoradiation  and signal transducer and activator of transcription 1, which is frequently found to be constitutively activated in a great variety of tumors including head and neck cancer and serve as appositive predictive marker.  As it is well known that tumor progression is angiogenesis dependent, we tried to explore the role of vascular endothelial growth factor (VEGF) marker of angiogenesis as a predictive marker. The research for such clinical and biological predictors may assist in the selection of patients likely to benefit from chemotherapy. Since there is only limited information about the expression of VEGF in oral cancer and its significance in evaluating the response to chemotherapy, we studied the expression of this angiogenic factor and elucidate its possible predictive role in patients with oral cancer treated with conventional chemotherapy.
| Materials and Methods|| |
Patients with biopsy proven locally advanced squamous cell oral cancer having borderline operability defined as disease involving the subcutaneous tissue of the cheek up to the zygoma, tumor extension above the level of sigmoid notch, extensive skin involvement not amenable for reconstruction, fixed N3 nodes, high infratemporal fossa extension (i.e., above the sigmoid notch) not amenable for primary surgery, or unfit to undergo surgery in the primary setting were included in the study after standard staging work-up. VEGF status was studied by immunohistochemistry with rabbit polyclonal antibody to VEGF (BioGenex Laboratories, USA) in biopsy tissue. Detection was done using polymer-based kit with horseradish peroxidase complex and three diaminobenzidine tetrahydrochloride as substrate. Five hundred squamous cells were counted in each case and brown staining of cells indicated positivity. Percent cell expression was done manually, and percent filled area expression was done by morphometry. 
The intensity of VEGF staining in 500 squamous cells of each case was calculated as below:
- Number of cells with negative VEGF expression ×0= A
- Number of cells with 1 (+) intensity ×1= B
- Number of cells with 2 (++) intensity ×2= C
- Number of cells with 3 (+++) intensity ×3= D.
VEGF score is the sum of the above. The minimum and maximum possible limits of the VEGF score were 0-1500. In addition, the mean VEGF score for each group was calculated. VEGF grading for all tumor tissues was done using the total VEGF score as below:
VEGF grade VEGF score:
- Grade 0: 0-50
- Grade 1: 51-500
- Grade 2: 501-1000
- Grade 3: 1001-1500.
Tissues with >50% positive cells were considered to have high VEGF expression.
Neoadjuvant chemotherapy (NACT) regimen comprising paclitaxel 180 mg/m 2 day 1, cisplatin 80 mg/m 2 day 2, and 5-fluorouracil 750 mg/m 2 day 1-5 was administered for three cycles before surgery or as definitive chemoradiotherapy. The patients were followed prospectively, and response to chemotherapy is assessed after three cycles and categorized according to the WHO criteria of tumor response.  VEGF score of the tumor was then correlated with chemotherapy response to study its role as a predictive marker.
In this study, the responders were divided into three groups, namely, complete, partial, and nonresponders. Partial responders include patients with partial and minimal response to chemotherapy, whereas nonresponders include patients with stable and progressive disease. Patients were then given further treatment, i.e., surgery if the disease is amenable for resection with negative margins with adjuvant radiation or definitive chemoradiation in those not fit to undergo surgery.
The statistical analysis was done using IBM Statistical package for social sciences (SPSS) (SPSS 15, Contractor/Manufacturer is SPSS Inc., 233, South Wecker Drive, 11 th Floor, Chicago, IL60606-6412). The values were represented in number (%) and mean ± standard deviation. Following statistical tests were used ANOVA test to compare within group and between group variances. Student's t-test is used to test the significance between two means.
| Results|| |
A total of forty patients with squamous cell carcinoma of the oral cavity were recruited and analyzed for (1) all forty patients were taken for estimation of VEGF status of cancer tissue and compare it with normal control mucosa and (2) 35 patients out of 40 received NACT and were included for studying the role of VEGF as a predictive marker. Therefore, for estimating VEGF expression in oral cancer, all forty patients were taken; and 35 patients with locally advanced cancer with borderline operability were included for studying the role of VEGF as a predictive marker. Majority (62.5%) were in the age range of 45-50 years. Thirty-three (82.5%) patients were males and 7 (17.5%) patients were females. Alveolo-buccal complex was the most common primary tumor site in our study, i.e., 26 (65%) patients. The corresponding VEGF scores for the various subsites and tumor morphology were also studied, and no association of VEGF score with the subsite or morphology was found. Seven (17.5%) patients had early stage oral cancer and 33 (83%) patients had locally advanced oral cancer (i.e., Stage III and IV). The mean VEGF score in early stage was 225, whereas in locally advanced stage, the score is 1111 which was statistically significant (P < 0.05). Majority of patients, i.e., 27 (67.5%) were of T4 stage. Three (7.5%) patients had T3 tumor, 8 (20%) patients had T2 tumors, and 2 (5%) patients had T1 tumors.
Vascular endothelial growth factor score
All the forty patients were VEGF-positive and had a score ranging from 60 to 1425 with a mean of 1023. Four (10%) had Grade 1 VEGF positivity (score 50-500), 10 (25%) had Grade 2 VEGF positivity (501-1000), and 26 (65%) had Grade 3 VEGF positivity. VEGF expression was more common in the cancerous tissue compared with the normal surrounding mucosa taken as control (mean VEGF score = 30). Thus, VEGF is overexpressed in cancerous mucosa as compared to noncancerous mucosa. Out of forty patients, 35 received neoadjuvant treatment according to a defined protocol as mentioned above and were analyzed for VEGF role as a predictive marker.
Postchemotherapy characteristics of study patients in group which received chemotherapy (n = 35)
Two patients had early stage tumors, i.e., Stage I and II (5.7%) and 33 patients (94.3%) had locally advanced tumor (Stage III and IV) before administration of chemotherapy, whereas after 3 cycles of chemotherapy, 7 (20%) patients had early stage and 27 (77%) had locally advanced tumor. One patient had a complete clinical response after administration of chemotherapy.
In patient who received NACT, majority of patients, i.e., 24 (68.5%) had T4 tumor, whereas 5 patients had T3 tumor, 5 had T2 tumor, and 1 had T1 tumor before chemotherapy administration. After administration of chemotherapy, 17 patients had T4 tumor, 14 patients had T2 tumor, 3 patients had T1 tumor, and 1 patient had complete disappearance of tumor after chemotherapy. No patient had residual T3 tumor after chemotherapy. Thus, administration of chemotherapy downstaged T4 tumor to T1 and T2 in 5 patients and 3 patients with T3 tumors is downstaged to T1 and T2.
Prechemotherapy 5 (14.3%) patients had no nodal involvement, whereas 3 (85.7%) patients had nodal involvement (N1, N2, and N3). After administration of chemotherapy, 12 (34.3%) patients had no palpable nodes, whereas 21 (65.7%) patients had node-positive disease, i.e., (N1 or N2) clinically.
Overall, one patient had complete response to treatment, 22 patients had less than complete response (partial response 12 patients, minimal response 10 patients), whereas 12 patients had no response or progressed on treatment. Mean VEGF score in complete responders was 425, partial responders (partial and minimal response) was 1049.7, and nonresponders (stable and progression) was 1156.82. Thus, in our study, responders had significantly lower VEGF score as compared to nonresponders to chemotherapy. It was seen that in patients showing no response to treatment, the mean total VEGF score was significantly higher as compared to those showing complete or partial response [Table 1]. Thus, VEGF score correlated inversely with chemotherapy response, but no significant association was seen between VEGF grade and chemotherapy response [Table 2].
|Table 1: Response in relation to vascular endothelial growth factor score|
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|Table 2: Response in relation to vascular endothelial growth factor grade|
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Twenty-nine patients out of forty underwent wide excision ± segmental/hemimandibulectomy and neck dissection. Five underwent surgery primarily without any neoadjuvant treatment. Twenty-five patients received adjuvant radiotherapy either due to advanced Stage III and IV, multiple positive nodes or positive margins or extracapsular extension. Six patients received definitive chemoradiation due to either progression of tumor or patient refused surgery after administration of chemotherapy. The remaining patients received radiation, as they remain inoperable after treatment.
| Discussion|| |
Oral cancer is the sixth most common cancer worldwide with an estimated 700,000 cases reported annually.  Oral cancer is more common among males with a male:female ratio of 2-3:1.  In Western countries, floor of mouth and tongue is the most common site, whereas in India, it is the alveolo-buccal complex.  Angiogenesis is essential for solid tumor growth and facilitates tumor progression and metastasis.  The development of tumor occurs in prevascular and vascular phase, regulated by multiple biochemical and genetic mechanisms such as basic fibroblast growth factor (FGF), transforming growth factor-beta, FGF, and VEGF. , Of all these factors, VEGF is considered the most important. VEGF family currently includes six members: VEGF-A, placenta growth factor, VEGF-B, VEGF-C, VEGF-D, and orf virus VEGF (called VEGF-E).  The founding member VEGF-A plays an essential role in angiogenesis and blood-borne metastasis.  VEGF-C appears to regulate lymphatic growth. VEGF-D acts on the same receptor as VEGF-C and has a similar role as VEGF-C. Expression of VEGF-C and VEGF-D was detected frequently in node positive compared to node-negative tumors. VEGF-A and VEGF-B are overexpressed in cancerous mucosa compared to adjacent normal appearing mucosal epithelium and normal mucosa. No significant association was found between VEGF expression and clinical staging in oral cancer. , However, some studies suggest that VEGF correlates with the stage of tumor.  The present study showed increased VEGF expression with advanced stage. Thus, VEGF expression with clinical stage is controversial, and no definite conclusion can be drawn.
In resectable oral cancer, the use of chemotherapy is controversial; some studies proposed the use of neoadjuvant chemoradiation, whereas others found that chemotherapy in combination with radiation led to significant improvement in local control and disease-free survival but not overall survival. , The administration of chemotherapy as neoadjuvant produced a response in 70-90% of patients. Published data correlating the degree of tumor microvessel density and clinical parameters in oral cancer is inconsistent. Tumor microvessel density may predict a favorable response to radiotherapy, particularly in nasopharyngeal carcinoma whether it predicts chemotherapy response is not studied.  Survival analysis failed to establish a prognostic significance of the marker.  Tumors with a greater count of microvessels are likely to present a more effective therapeutic response as microvascularization and blood flow within a tumor, determine not only the growth and metastasis but also tissue access to anticancer drugs. The tumor vascularization and expression of VEGF have been explored for its potential role as a factor that might affect the response to chemotherapy and radiotherapy.  Such relation was found between vascular density and response to combination chemotherapy of squamous cell head and neck cancer and to doxorubicin of nonsmall cell lung cancer.  VEGF overexpression is associated with an aggressive phenotype, i.e., lower degree of differentiation and minimal response and resistance to chemotherapy or radiotherapy.  The assessment of VEGF expression in pretreatment biopsy specimen could provide additional information to identify patients with a poor chance of response to NACT. , It has also been seen that response rate of 72% to NACT may translate 74% patients initially inoperable to become operable, improving the outcome. It has been observed that in resectable oral cancer Stage III and IV, primary chemotherapy seem to play a role in reducing the number of patients who needed to undergo mandibulectomy.  In our study, responders to chemotherapy had significantly lower VEGF score as compared to nonresponders (VEGF 1156.82) (P = 0.019).
In the present study, the tumors with lesser degree of differentiation showed less response to chemotherapy. VEGF expression is associated with an aggressive phenotype, i.e., lower degree of differentiation and minimal response to chemotherapy. At this stage, however the use of VEGF as a predictor of measurement of tumor progression remains elementary and further research to clarify its usefulness is required. Therefore, further studies with more uniform patient population, large sample size, and quantitative methods are needed to resolve this finding.
| Conclusion|| |
In this small study, we studied the expression of VEGF in oral cancer and tried to explore the predictive role of VEGF and found that patients with increased VEGF had decreased response to chemotherapy. We can conclude from this study that VEGF is significantly overexpressed in oral cancer, and overexpression of VEGF was found to be associated with chemoresistance. These initial encouraging results in this small study need to be confirmed by a larger study to establish the role of VEGF as an important predictor of response to chemotherapy.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]